Evaluation of integrin αvβ3-targeted imaging for predicting disease progression in patients with high-risk differentiated thyroid cancer (using 99mTc-3PRGD2)

Background High-risk differentiated thyroid cancer (DTC) needs effective early prediction tools to improving clinical management and prognosis. This cohort study aimed to investigate the prognostic impact of 99mTc-PEG4-E[PEG4-c(RGDfK)]2 (99mTc-3PRGD2) SPECT/CT in high-risk DTC patients after initial radioactive iodine (RAI) therapy. Methods Thirty-three patients with high-risk DTC were prospectively recruited; all patients underwent total thyroidectomy and received 99mTc-3PRGD2 SPECT/CT before RAI ablation. Follow-up was done with serological and imaging studies. The correlation between 99mTc-3PRGD2 avidity and remission rate for initial RAI therapy was evaluated using logistic regression analysis. The prognostic value of 99mTc-3PRGD2 SPECT/CT was evaluated by Kaplan-Meier curve and Cox regression analysis. Results 99mTc-3PRGD2 avidity was significantly correlated with the efficacy of initial RAI ablation and an effective predictor for non-remission in high-risk DTC (OR = 9.36; 95% CI = 1.10–79.83; P = 0.041). 99mTc-3PRGD2 avidity was associated with poor prognosis in patients with high-risk DTC and an independent prognostic factor for shorter progression-free survival (PFS) (HR = 9.47; 95% CI = 1.08–83.20; P = 0.043). Survival analysis, which was performed between DTC patients with concordant (131I positive/99mTc-3PRGD2 positive) and discordant (131I negative/99mTc-3PRGD2 positive) lesions, indicated that patients with concordant lesions had significantly better PFS than those with discordant lesions (P = 0.022). Moreover, compared with repeated RAI, additional surgery or targeted therapy with multikinase inhibitors could lead to a higher rate of remission in 99mTc-3PRGD2-positive patients with progressive disease. Conclusions 99mTc-3PRGD2 SPECT/CT is a useful modality in predicting progression of the disease after initial RAI and guiding further treatment in high-risk DTC patients.


Introduction
The incidence of thyroid cancer continuously increased over the past three decades [1]. Differentiated thyroid cancer (DTC) accounts for more than 90% of all thyroid cancers, and prognosis in the majority of DTC patients is excellent. However, increasing cases, especially those with high-risk DTC, have been reported to develop local recurrence or metastatic disease after initial surgery and radioactive iodine (RAI) ablation [2]. Two-thirds of these patients will never be cured with RAI therapy and become RAI-refractory (RAIR), with a 3-year survival rate of less than 50% [3]. Early identification of the propensity for disease progression after initial therapy in high-risk DTC patients can assist physicians to develop prompt and individualized treatment plans.
The routine evaluation of DTC patients includes the measurement of serum thyroglobulin (Tg), 131 I scintigraphy, ultrasound, and computed tomography (CT). Due to positive thyroglobulin antibody (TgAb) or undifferentiated lesions that do not secrete Tg, serum Tg may not be a reliable predictor in some patients. 131 I scintigraphy also often fail to detect lesions with impaired ability to concentrate iodine. CT and ultrasound provide only anatomic data, which may lag behind functional changes. Hence, 18 F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is gradually being used to localize lesions in patients with suspected RAIR-DTC. However, due to the common co-existence of iodine-sensitive and -refractory disease in high-risk DTC, the relatively low glucose metabolism in the lesions with heterogeneous cells are likely to be missed on FDG PET/CT [4]. Moreover, enhanced glucose uptake in inflammatory tissues, such as reactive lymph nodes, reduces the specificity of 18 F-FDG PET/ CT [5,6]. A more effective imaging method is needed for early detection of advanced diseases in high-risk DTC patients.
Integrin α v β 3 , which is significantly upregulated on several tumor cells and activated endothelial cells, plays essential roles in neoangiogenesis and tumor progression as a member of the arginine-glycine-aspartate (RGD)binding subfamily [7]. Unlike 18 F-FDG PET/CT, which is a diagnosis-only modality, RGD imaging provides not only a specific method for visualizing tumor angiogenesis but also therapeutic implications for antiangiogenetic and anti-α v β 3 drugs [8][9][10]. 99m Tc-PEG 4 -E[PEG 4 -c(RGDfK)] 2 ( 99m Tc-3PRGD 2 ), a novel RGD peptide tracer, is specifically designed to recognize integrin α v β 3 . 99m Tc-3PRGD 2 has been used to trace primary or metastatic lesions in patients with various tumors, including lung, breast, esophageal and thyroid cancers [11][12][13][14]. Our previous studies have validated 99m Tc-3PRGD2 was a valuable probe for the detection of recurrent lesions with negative radioiodine whole-body scintigraphy (WBS) [15]. In addition, integrin α v β 3 has been reported to interact with the vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor (PDGFR) [16,17]. The cross-talk between integrin α v β 3 and VEGFR-2/PDGFR is crucial for endothelial cell activation and angiogenesis. In vivo studies demonstrated that 99m Tc-3PRGD 2 imaging was a noninvasive tool to predict and evaluate the response to therapy with antiangiogenic agents in breast cancer [8].
In the present study, for the first time, we analyze the utility of 99m Tc-3PRGD 2 single photon emission computed tomography/computed tomography (SPECT/CT) for the prognostication of therapeutic effect and disease progression in patients with high-risk DTC after initial RAI ablation.

Patients
A total of 33 patients with high-risk DTC being managed in the Department of Nuclear Medicine at the First Affiliated Hospital of Xi'an Jiaotong University between May 2017 and December 2020 were enrolled in this study. The cases should meet the following inclusion criteria: (i) patients who underwent total thyroidectomy and were prepared for RAI, (ii) histologically confirmed DTC (papillary and follicular thyroid carcinoma), (iii) defined as high-risk disease according to the 2015 American Thyroid Association (ATA) guidelines [18], that is the patients with gross extrathyroidal extension (ETE), incomplete tumor resection, distant metastases, postoperative serum Tg suggestive of distant metastases, pathologic N1 disease with any metastatic lymph node ≥3 cm in largest dimension, or follicular thyroid carcinoma with extensive vascular invasion (more than 4 foci of vascular invasion). The demographic parameters, including surgical summary and histopathology reports, of all the study participants were recorded. Serum Tg, TgAb and TSH values were measured by radioimmunoassay method. This study was approved by the ethic committee of the First Affiliated Hospital of Xi'an Jiaotong University. Written informed consent was obtained from all patients prior to their enrolment. This study has been registered at http:// www. chictr. org. cn/ (No. ChiCTR1900028095).

99m
Tc-3PRGD 2 SPECT/CT imaging 99m Tc-3PRGD 2 SPECT/CT and planar imaging were performed at 1 month after surgery and 1 to 3 days before RAI treatment using a dual-head gamma-camera (Discovery NM/CT 670 pro; GE Healthcare, Milwaukee, USA) with low-energy high-resolution collimators and a 20% energy window centered on 140 keV. The SPECT and coregistered spiral CT were performed at 40-60 min post injection of 740-1110 MBq (20-30 mCi) of 99m Tc-3PRGD 2 . SPECT scan (matrix 128 × 128 pixels, zoom 1.0, 30 s/frame/6°) of the neck and chest was performed with the patients' arms raised above their head, followed by CT scan (120 kV, 160 mAs) with the same range of SPECT. The speed of whole-body planar scan (matrix 256 × 1024 pixels) was set at 18 cm/min. The imaging of each patient was reconstructed and analyzed using Xeleris 3.0 workstation (GE Healthcare).

Image analysis and interpretation
The 99m Tc-3PRGD 2 images were independently reviewed by 2 experienced nuclear medicine physicians who were blinded to the source, history and pathologic conditions of the patients. A positive lesion was defined as the uptake of radiotracer above its background, excluding physiologic uptake [7,15]. Disease foci were divided into the following regions: thyroid bed (remnant/recurrent), nodal disease (cervical or mediastinal) and lung lesions. The number of lesions at each site was recorded, except in the lungs. When more than 5 lesions were detected in lungs, 5 lesions with the highest uptake were selected from each case for further analysis [4]. The tumor-tobackground (T/B) ratio of the positive lesions on SPECT was measured and calculated by the same person using a consistent standard. Briefly, regions of interest (ROI) were drawn around the lesions with reference to integrated CT. And on the same section, a background ROI was set in the surrounding normal soft tissue. The T/B ratio was calculated by dividing the mean count of tumor ROI by the mean count of background ROI. In addition, the tumor volume of interest (VOI) for each lesion was calculated, and maximum standardized uptake value (SUV max ) was defined as the maximum concentration in the target lesion (maximum radioactivity/volume of VOI))/(injected radioactivity/body weight). The detailed calculation of SUV max was based on a patented algorithm (Patent No.: US11189374B2) [19].

Follow-up and treatment intervention
After initial RAI and post-therapeutic WBS, the patients were followed up for an average of 21 months. Levothyroxine was administrated to all the patients to suppress serum TSH levels. The ultrasound of neck and unstimulated thyroglobulin (T4-Tg) level were examined every 3 to 6 months during follow-up visits. Additional CT and/ or magnetic resonance imaging (MRI) was performed every 3 to 6 months in patients who demonstrated distant metastasis. Additional therapies like secondary surgery, RAI, or targeted therapy were recorded.
Based on the radiologic findings and serum Tg levels, disease status was classified as complete cure, clinical improvement, stable disease, or progressive disease [20][21][22]. Complete cure was defined as undetectable TSHstimulated Tg levels (or < 2.0 μg/L) and in the absence of TgAb with no evidence of structural disease on imaging. Clinical improvement was defined as at least 25% reduction in serum Tg levels with ≥30% decrease in the cumulative diameter of lesions. Progressive disease was defined as an increase of at least 25% in the serum Tg levels, with > 20% increase in the sum of lesion diameters or 5 mm increase in the sum of lesion diameters or appearance of new lesions. Stable disease was defined as < 25% increase or decrease in the serum Tg level with no obvious change in the cumulative diameter of lesions (neither sufficient shrinkage for clinical improvement nor sufficient increase for progressive disease).

Statistical analysis
Patients who achieved complete cure and clinical improvement were categorized to the remission group, while those with stable and progressive disease were categorized to the non-remission group. Multivariate logistic regression model was used to investigate factors that associated with non-remission. Progression-free survival (PFS), defined as the time interval from the date of initial RAI to the date of disease progression, was the primary end point of this study. Survival data were estimated by Kaplan-Meier analysis with the log-rank test, and Cox regression analyses for PFS were used to identify the significant prognostic factors in high-risk DTC. For patients with multiple 99m Tc-3PRGD 2 -positive lesions, the median of T/B ratio and SUV max values was calculated to perform survival analysis based on semi-quantitative SPECT/CT parameters. A P values of < 0.05 were considered statistically significant on the basis of 2-sided testing. All statistical analyses were performed using SPSS 22.0 and GraphPad Prism v5.0.
Kaplan-Meier survival curves revealed that 99m Tc-3PRGD 2 uptake inversely correlated to PFS of high-risk DTC patients (P = 0.035; Fig. 1A). The median PFS of patients with 99m Tc-3PRGD 2 non-avidity was not reached versus 19 months in patients with 99m Tc-3PRGD 2 avidity. Multivariate Cox regression analysis showed that 99m Tc-3PRGD 2 avidity was significantly associated with shorter PFS (HR = 9.47; 95% CI = 1.08-83.20; P = 0.043; Table 3). These results suggested that 99m Tc-3PRGD 2 avidity was an independent risk factors for progression in high-risk DTC patients. Moreover, of the 25 patients with 99m Tc-3PRGD 2 -positive lesions, 9 patients showed 131 I uptake in the lesions on the 131 I post therapy scan, while 16 had no 131 I uptake in the lesions. Figure 1B   The Kaplan-Meier method was used to estimate PFS probabilities based on whether T/B ratio or SUV max was greater than, or less than median for each variable. There was a trend for improved PFS with T/B ratio or SUV max less than the median (Fig. 1C and D). The median PFS of patients with T/B ratio greater than median was 14 months versus 21 months in patiens with T/B ratio less than median. In patients with SUV max greater than median, the median PFS was 12 months versus 21 months in patients with SUV max less than median. However, this trend did not reach the threshold of statistical

Managements of patients with 99m Tc-3PRGD 2 -positive lesions and progressive disease
After initial surgery and RAI ablation, 14 patients with 99m Tc-3PRGD 2 -positive lesions had progressive disease during follow-up. Of the 14 patients, 10 patients underwent additional surgery of the neck or lung because of lymph node or lung metastases, and malignant diseases were pathologically validated in resected lesions in 9 patients. As shown in Table 5, the result of the follow-up after additional surgery was remission in 6/10 patients, stable disease in 1/10 patients and progressive disease in 3/10 patients. Two cases received another RAI treatment. At the end of follow-up, progressive disease was observed in both patients who received second RAI. Representative 131 I post-therapeutic WBS and 99m Tc-3PRGD 2 SPECT/CT images of 1 of these 2 patients were showed in Fig. 3. Targeted therapy with multikinase inhibitors (MKIs) was applied in 2 patients. Of the 2 patients, one patient achieved clinical improvement, while another patient lost to follow-up.

Discussion
Thyroid cancer is clinically heterogeneous, varying from indolent to aggressively proliferative disease. Patients with high-risk DTC, which often represents less welldifferentiated disease, have a lower chance of response to RAI therapy than low-risk patients [23]. More sensitive imaging modalities for identification of aggressive status is critical to the prognosis of high-risk DTC patients. RGD-peptide based SPECT/CT is a neo-angiogenesis imaging modality which has high affinity and specificity towards integrin α v β 3 . Xu et al. reported that 99m Tc-Galacto-RGD 2 SPECT/CT had higher sensitivity than 131 I WBS and morphological imaging in the detection of lymphatic and bone metastasis in DTC patients [7]. The overall sensitivity and specificity of 99m Tc-Galacto-RGD 2 SPECT/CT were 92.86 and 86.36%, respectively, in the detection of metastatic DTC diseases. In our previous study, 99m Tc-3PRGD 2 SPECT/CT showed high sensitivity in the detection of recurrence among DTC patients with Tg elevation but negative iodine scintigraphy (TENIS), and the sensitivity was improved to 100% in patients with TSH-stimulated Tg > 30 ng/mL [15]. However, there are few studies on the ability of RGD-based imaging to predict the prognosis after initial surgery and RAI in DTC patients. In this study, we found that 99m Tc-3PRGD 2 avidity was an effective predictor for non-remission in high-risk DTC. The present study also found that 99m Tc-3PRGD 2 avidity was associated with poor prognosis in patients with high-risk DTC. 99m Tc-3PRGD 2 avidity was significantly correlated with PFS in multivariate analysis, which indicated 99m Tc-3PRGD 2 avidity as an independent risk indicator for PFS in high-risk DTC.
In this study, about three-fourth of all patients (75.8%) had 99m Tc-3PRGD 2 -positive lesions at initial diagnosis before RAI treatment. Of the 25 patients with 99m Tc-3PRGD 2 -positive lesions, about two-third of patients (64.0%) had no 131 I uptake in the lesions on 131 I post therapy WBS, which would have been missed by standard RAI alone. Matching iodine-and 99m Tc-3PRGD 2 -positive lesions were observed in 9 patients. Survival analysis indicated that the presence of 99m Tc-3PRGD 2 uptake in tumor lesions and the absence of 131 I uptake in these lesions were significantly related to a worse PFS after initial RAI ablation. The role of Abbreviations: HR, hazard ratio; T/B ratio: tumor-to-background ratio; SUV max : maximum standardized uptake value 99m Tc-3PRGD 2 SPECT/CT in therapy management of high-risk DTC was further observed in 14 99m Tc-3PRGD 2 -positive patients having progressive disease after initial surgery and RAI. We found that additional surgery or MKIs therapy might lead to a higher rate of remission than repeated RAI in patients with 99m Tc-3PRGD 2 -positive lesions. Our results strongly suggested a linking between 99m Tc-3PRGD 2 avidity and radioiodine refractory disease. This finding is consistent with prior studies by Zhao et al., which showed that RAIR metastatic lesions can be traced using 99m Tc-3PRGD 2 SPECT imaging [14].
Nowadays, 18 F-FDG PET/CT is the main method recommended by the ATA guidelines for the detection of RAIR-DTC. Many studies have demonstrated its utility in the detection of structural disease in RAIR-DTC patients with increasing sensitivity at higher levels of serum Tg, and changes of intermediate or high-risk patient management [24][25][26]. However, there are still some RAIR-DTC patients who have a negative 18 F-FDG PET/CT, which drives the need for alternative imaging modalities. The PET or SPECT imaging of integrin α v β 3 has recently been evaluated in refractory DTC, and some studies found that RGD-based imaging has better diagnostic performance than 18 F-FDG [4,27]. For instance, Parihar et al. reported 68 Ga-DOTA-RGD 2 PET/CT had higher specificity and overall accuracy than 18 F-FDG PET/CT in detection of lesions in RAIR-DTC patients [4]. They noted that 68 Ga-DOTA-RGD 2 PET/CT and 18 F-FDG PET/CT showed a similar sensitivity of 82.3%, however 68 Ga-DOTA-RGD 2 PET/CT had a higher specificity of 100% compared to 50% on 18 F-FDG PET/CT. Our study indicated that compared with repeated RAI, additional surgery or targeted therapy with MKIs could lead to a higher rate of complete or partial remission in 99m Tc-3PRGD 2 -positive patients, suggesting 99m Tc-3PRGD 2 scan could be able to guide the adjustments in management after the initial surgery and RAI ablation. Considering that the association of 99m Tc-3PRGD 2 avidity with unfavorable prognosis, patients with a positive 99m Tc-3PRGD 2 scan should be asked for a closer followup to detect recurrent or metastatic diseases in a timely manner. Therefore, 99m Tc-3PRGD 2 SPECT/CT is a valuable diagnostic method for high-risk DTC patients and an effectively complementary modality for 18 F-FDG PET/ CT in refractory DTC.
In our study, further survival analysis revealed a trend towards worse PFS in patients with higher than median values for T/B ratio and SUV max . Recent studies reported the T/B ratios of 99m Tc-3PRGD 2 in metastatic DTC lesions were positively correlated with growth rates of these lesions [14] and patients' clinical stages [7]. Another study demonstrated that the SUV max of RAIR-DTC lesions on 68 Ga-DOTA-RGD 2 PET/CT had a strong positive correlation with serum TSH-stimulated Tg levels, which reflecting the disease burden [4]. Hence, the parameters of RGD uptake could be used as potential imaging biomarkers for tumor burden and biologic aggressiveness. We did not detect a lineal correlation between Tg levels and 99m Tc-3PRGD 2 uptake in this study. The possible reason is that the serum Tg was tested before initial RAI treatment, so that Tg was partly secreted by residual thyroid tissue, which could not reflect the real burden of recurrent or metastatic diseases.
In this study, the DTC patients with positive 99m Tc-3PRGD 2 lesions all received full TSH suppression of less than 0.1 mIU/l with levothyroxine, but still had a high rate of disease progression. Thyroid hormone has been reported to increase tumor growth in various types of cancer, including hepatocellular, colorectal, and lung cancers [28][29][30]. A retrospective study followed 867 patients with intermediate-and high-risk DTC for a median of 7 years, documenting that patients with suppressed TSH levels were associated with worse 3-year overall survival  [31]. The widespread use of TSH-suppressive therapy has recently been questioned, and individualized treatment based on each patient's characteristics has been proposed. More recently, it was reported that integrin α v β 3 has a high affinity binding site for thyroxine [32]. Thyroxine has been suggested to promote proliferation and angiogenesis in multiple cancer types via binding with integrin α v β 3 [33,34]. Therefore, TSH suppressive doses of levothyroxine in DTC patients with tumoral integrin α v β 3 expression may have to be reconsidered, and integrin α v β 3 -targeting 99m Tc-3PRGD 2 SPECT/CT could theoretically be utilized to identify these patients. Further mechanistic and clinical studies are needed to test this hypothesis.
There are still some limitations in this study. First, the follow-up was relatively short. Further studies with larger cohort of patients and longer follow-up period are required to validate our findings. Second, only 1 patient in this study Fig. 2 Representative 99m Tc-3PRGD 2 SPECT/CT images of patients with 131 I negative and 99m Tc-3PRGD 2 positive lesions. A 99m Tc-3PRGD 2 SPECT/ CT detected lung metastases in a 63-year-old female PTC patient with initial TSH-stimulated Tg 3.1 ng/ml and positive TgAb. 99m Tc-3PRGD 2 SPECT/ CT showed a focal uptake in the right pulmonary nodule. B 131 I post therapy WBS showed no uptake in this pulmonary nodule. C 99m Tc-3PRGD 2 SPECT/CT detected lymph node metastases in a 41-year-old female PTC patient with initial TSH-stimulated Tg > 500 ng/ml. 99m Tc-3PRGD 2 SPECT/ CT showed focal uptakes in left cervical lymph nodes (level II and IV). The diffuse 99m Tc-3PRGD 2 accumulation in thyroid bed was considered to be caused by postsurgical reactions. D 131 I post therapy WBS showed no uptake in these lymph nodes. These neck lymph nodes were positive for metastatic PTC on histopathologic examination after secondary surgery Table 4 Sites of lesion detection and radiotracer uptake parameters on 99m Tc-3PRGD 2 SPECT/CT Abbreviations: T/B ratio tumor-to-background ratio, SUV max maximum standardized uptake value  18 F-FDG PET/CT and 99m Tc-3PRGD 2 SPECT/CT images, which was pathologically validated by fine needle aspiration biopsy. Future prospective research including parallel 18 F-FDG PET/CT and 99m Tc-3PRGD 2 SPECT/CT examinations is needed to clarify the effect of FDG PET/ CT results on prognostic significance of 99m Tc-3PRGD 2 SPECT/CT and compare the diagnostic and prognostic values of the 2 imaging modalities. Third, further experiments are indispensable to determine the underlying mechanism of integrin α v β 3 promoting DTC. Table 5 The management of patients with 99m Tc-3PRGD 2 positive lesions and progressive disease after initial therapy and the results of the last follow-up Abbreviations: RAI radioactive iodine Tc-3PRGD 2 SPECT/CT before RAI showed a focal uptake in the left thyroid bed in planar image (A) and transaxial fused SPECT/CT (B), which was seen a soft-tissue mass on CT image. 131 I post therapy WBS found a slight radioiodine uptake in planar image (D) and transaxial fused SPECT/CT (E) in the right thyroid bed. The lesion in the left thyroid bed was negative on 131 I WBS (F). 99m Tc-3PRGD 2 SPECT/CT showed no 99m Tc-3PRGD 2 uptake in the right thyroid bed (C). Four months after initial RAI, the serum TSH-stimulated Tg of the patient elevated to 734 ng/ml. The neck ultrasound examination also detected local recurrence in the left thyroid bed. The patient received another RAI, and the post-therapeutic WBS showed only a vague uptake of radioiodine in the right thyroid bed (G, H). The lesion in the left thyroid bed was still negative on secondary 131 I WBS (I)

Conclusion
In this study, 99m Tc-3PRGD 2 SPECT/CT was found to be a promising predictor for poor therapeutic effect and unfavorable prognosis after initial RAI treatment in patients with high-risk DTC. This imaging modality also contributed to the selection of therapy strategies and could been established as a standard procedure in the treatment of high-risk DTC patients.